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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0634—Cells from the blood or the immune system
  • C12N5/0646—Natural killers cells [NK], NKT cells
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  • A61K40/00—Cellular immunotherapy
  • A61K40/10—Cellular immunotherapy characterised by the cell type used
  • A61K40/15—Natural-killer [NK] cells; Natural-killer T [NKT] cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
  • A61K40/41—Vertebrate antigens
  • A61K40/42—Cancer antigens
• • A—HUMAN NECESSITIES
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  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2121/00—Preparations for use in therapy
• • A—HUMAN NECESSITIES
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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  • C12N2500/00—Specific components of cell culture medium
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  • C12N2500/00—Specific components of cell culture medium
  • C12N2500/50—Soluble polymers, e.g. polyethyleneglycol [PEG]
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/20—Cytokines; Chemokines
  • C12N2501/23—Interleukins [IL]
  • C12N2501/2302—Interleukin-2 (IL-2)
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  • C12N2501/30—Hormones
  • C12N2501/33—Insulin

Definitions

• Natural killer (NK) ceils are cytotoxic lymphocytes that constitute a major component of the innate immune system. NK cells, generally representing about 10-15% of circulating lymphocytes, bind and kill targeted ceils, including virus-infected ceils and many malignant cells, non-specifically with regard to antigen and without prior immune sensitization. Herberman et al., Science 214:24 (1981). Killing of targeted cells occurs by inducing cell lysis. NK cells used for this purpose are isolated from the peripheral blood lymphocyte (“PBL”) fraction of blood from the subject, expanded in cell culture in order to obtain sufficient numbers of celis, and then re-infused into the subject. NK cells have been shown to be somewhat effective in both ex vivo therapy and in vivo treatment. However, such therapy is complicated by the fact that not all NK cells are cytolytic and the therapy is specific to the treated patient.
• PBL peripheral blood lymphocyte
• NK-92 ® cells have previously been evaluated as a therapeutic agent in the treatment of certain cancers.
• the use of NK-92 ® cells m therapeutic applications requires consistent quantities and qualities between batches thus many of the media which use animal components such as serum are unsuitable for this purpose.
• certain media that have defined chemical compositions can be used, these media were specifically designed to support the growth of other types of cells, such as lymphokine activated killer cells.
• This type of media may contain unnecessary components that increase the costs of production. This type of media may also leave process residuals that affect the efficacy and purity- of the NK-92 ®3 cells.
• a need remains for an economical growth medium that is customarily designed for growing NK-92 ® cells.
• the medium composition comprises a basal medium supplemented one or more supplements selected from the group consisting of ethanolamine (or a derivative thereof), insulin, transferrin, and human albumin (HA).
• the basal medium is supplemented with sodium selenite and glucose.
• the basal medium itself comprises inorganic salts, vitamins and amino acids.
• the basal medium itself comprises sodium selenite and glucose.
• a method of culturing NK-92 ® cells comprising culturing NK-92 ® cells in a customized NK-92 ® culture medium comprising a basal medium and one or more supplements, and the one or more supplements comprise ethanolamine, an ethano!amine derivative, insulin, transferrin, sodium selenite, HA, or a combination thereof.
• the basal medium comprises inorganic salts, vitamins and amino acids.
• Also provided herein is a method of culturing NK-92 ® ceils comprising culturing NK- 92 ® cells in a customized NK-92 ® culture medium comprising a basal medium and one or more supplements, and the one or more supplements comprise ethanolamine, an ethanolamine derivative, or a combination thereof.
• the basal medium comprises inorganic salts, vitamins and amino acids.
• the ethanolamine derivative is an ethanolamide.
• the ethanolamide may be vaceemc acid ethanolamide, or oleic acid ethanolamide, palmitic acid ethanolamide, or stearic acid ethanolamide.
• the ethanolamine derivative is phosphatidylethanolamine.
• the one or more supplements comprise 1-7% human AB serum.
• the one or more supplements further comprise insulin, transferrin, selenium, or a combination thereof.
• the one or more supplements further comprise 300-600 lU/mL interleukin-2
• the one or more supplements further comprise 0.01% to 0.1% of poloxamer 188.
• the NK-92 ® cells cultured in the customized NK-92 ® culture medium have substantially the same or higher cytotoxicity', growth rate, and viability' compared to NK-92 ® cells grown in a reference growth medium.
• the NK-92 ® ceils cultured in the customized NK-92 ® culture medium have 85-100% viability.
• the NK-92 ® ceils cultured in the customized NK-92 ® culture medium show a direct cytotoxicity of and/or an ADCC of 60-100% at an effector to target ratio of 10: 1.
• the NK-92 ® cells cultured in the customized NK-92 ® culture medium have a doubling time of 30-50 hours.
• the NK-92 ® cells express a cytokine, Fc Receptor, a chimeric antigen receptor, or a combination thereof.
• the customized NK-92 ® culture medium comprises 0.05-40 mg/L of ethanolamine, an ethanolamme derivative, or a combination thereof.
• the customized NK-92 ® culture medium 4.5-20 g/L of glucose.
• the basal medium is further supplemented with 0.05% to 1.0% HA.
• the volume of the customized NK-92 ® culture medium is at least 5 liters.
• a cell culture comprising NK-92 ® cells in a customized NK-92 ® culture medium comprising a basal medium and one or more supplements, and the one or more supplements comprise ethanolamine, an ethanolamine derivative, insulin, transferrin, sodium selenite, HA, or a combination thereof, wherein the basal medium comprises inorganic salts, vitamins and ammo acids.
• a cell culture composing NK-92 ® cells in a customized NK-92 ® culture medium comprising a basal medium and one or more supplements, wherein the one or more supplements comprise ethanolamine, an ethanolamine derivative, or a combination thereof; and wherem the basal medium comprises inorganic salts, vitamins and ammo acids.
• the cell culture medium is at least 5 liters.
• the ethanolamine derivative is ethanolamide, wherein the ethanolamide is cis-vaccemc acid ethanolamide or oleic acid ethanolamide.
• the one or more supplements further comprise insulin, transferrin, selenium, or a combination thereof.
• the one or more supplements further comprise 0.01% to 0.1% of poloxamer 188.
• the customized NK-92 ® culture medium comprises 4.5-20 g/L glucose.
• the one or more supplements comprise 0.05% to 1.0% HA.
• the NK-92 ® cells express a cytokine, Fc Receptor, a chimeric antigen receptor, or a combination thereof.
• the basal medium comprises insulin, transferrin, selenium, or a combination thereof.
• the NK-92 ® cells have substantially the same or higher cytotoxicity, growth rate, and/or viability as compared to control NK-92 ® cells.
• the NK-92 ® cells that have been cultured in the customized NK-92 ® culture medium have 85-100% viability.
• the NK-92 ® cells that have been cultured in the customized NK-92 ® culture medium have a doubling time of 30-50 hours.
• the NK-92 ® ceils show a direct cytotoxicity and/or an ADCC of 60-100% when using an effector: target ratio of 10: 1.
• the NK-92 ® ceils maintain substantially the same viability and/or cytotoxicity after the they are crypreserved and thawed.
• Figure 1 is a graph showing the direct cytotoxicity of haNK ® cells against K562 target cells at growth cycle 5.
• the medium composition comprises a basal medium supplemented with one or more supplements comprising ethanolamine, an ethanolamine derivative, insulin, transferrin, sodium selenite, has, or combination thereof.
• the basal medium itself may comprise inorganic salts, vitamins and amino acids.
• a percentage when denoting a concentration, refer to a w/v percentage.
• 1.0% HA refers to the 1.0% w/v HA.
• concentrations in this disclosure refer to the final working concentration in the customized NK-92 ® culture medium.
• ail language such as“up to,”“at least,” “greater than,”“less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above.
• a range includes each individual member.
• a group having 1 -3 cells refers to groups having 1 , 2, or 3 cells.
• a group having 1 -5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.
• compositions and methods include the recited elements, but not excluding others.
• Consisting essentially of when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination. For example, a composition consisting essentially of the elements as defined herein would not exclude other elements that do not materially affect the basic and novel charactenstic(s) of the claims.“Consisting of’ shall mean excluding more than trace amount of other ingredients and substantial method steps. Embodiments defined by each of these transition terms are within the scope of the disclosure.
• NK cells are cells of the immune system that kill target ceils in the absence of a specific antigenic stimulus, and without restriction according to major histocompatibility complex (MHC) class.
• Target cells may be cancer or tumor cells.
• NK cells are characterized by the presence of CD56 and the absence of CDS surface markers.
• NK-92 ® or “NK92” is intended to refer to the original NK-92 ® cell lines as well as NK-92 ® cell lines, clones of NK-92 ® cells, and NK-92 ® cells that have been modified (e.g., by introduction of exogenous genes).
• NK-92 ® cells and exemplar ⁇ ' and non-limiting modifications thereof are described in U.S. Patent Nos. 7,618,817; 8,034,332; 8,313,943; 9,181 ,322; 9,150,636; and published U.S. Application No.
• NK-92 ® cells are known to persons of ordinary skill in the art, to whom such cells are readily available from NantKwest, Inc..
• aNKTM cells refers to the parental NK-92 ® cells. aNKTM cells depends on IL-2 for growth.
• haNK ® cells refers to NK-92 ® cells that have been engineered to express Fc receptor.
• taNK ® cells refers to NK-92 ® cells that have been engineered to express a chimeric antigen receptor (CAR) with affinity for a cancer specific antigen, a cancer associated antigen, or a tumor specific antigen.
• the tumor specific antigen is KER-2, e.g., human HER-2, and these NK-92 ® cells are referred to as HER-2 taNK ® cells.
• t-haNK ® cells refers to NK-92 ® cells that have been engineered to express an Fc receptor and a chimeric antigen receptor (CAR) with affinity for a cancer specific antigen, a cancer associated antigen, or a tumor specific antigen.
• the tumor specific antigen is CD 19, and these NK-92 ® cells are referred to as CD19 t-haNK ® cells.
• Fc receptor refers to a protein found on the surface of certain cells (e.g., natural killer cells) that contribute to the protective functions of the immune ceils by binding to part of an antibody known as the Fc region. Binding of the Fc region of an antibody to the Fc receptor (FcR) of a cell stimulates phagocytic or cytotoxic activity of a cell via antibody- mediated phagocytosis or antibody-dependent cell-mediated cytotoxicity (ADCC). FcRs are classified based on the type of antibody they recognize. For example, Fc- gamma receptors (FcyR) bind to the IgG class of antibodies.
• FcyR Fc- gamma receptors
• FcyRIH-A (also called CD 16) is a low r affinity Fc receptor bind to IgG antibodies and activate ADCC.
• FcyRHI-A are typically found on NK cells.
• NK-92 ® cells do not express FeyRIII- A.
• CAR chimeric antigen receptor
• the extracellular antigen- binding domain is a scFv that is specific for an antigen found on a cell of interest.
• a CAR- expressing NK-92 ® cell is targeted to cells expressing certain antigens on the cell surface, based on the specificity of the scFv domain.
• the scFv domain can be engineered to recognize any antigen, including tumor-specific antigens.
• Polynucleotides can have any three- dimensional structure and may perform any function, known or unknown.
• a gene or gene fragment for example, a probe, primer, EST or SAGE tag
• exons introns
• messenger RNA messenger RNA
• transfer RNA transfer RNA
• ribosomal RNA ribozymes
• cDNA recombinant polynucleotides
• branched polynucleotides plasmids
• a polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide.
• the sequence of nucleotides can be interrupted by non-nucleotide components.
• a polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component.
• the term also refers to both double- and single-stranded molecules. Unless otherwise specified or required, a polynucleotide encompasses both the double- stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.
• the term“expression” refers to the production of a gene product.
• the term“transient” when referred to expression means a polynucleotide is not incorporated into the genome of the cell.
• cytokine refers to the general class of biological molecules which effect cells of the immune system.
• exemplary cytokines include, but are not limited to, interferons and interleukins (IL), in particular IL-2, IL-12, IL-15, IL-18 and IL-21.
• IL-2 interferons and interleukins
• IL-12 interleukins
• IL-15 interleukins
• IL-18 interleukins
• IL-21 interleukins
• the cytokine is IL-2.
• vector refers to a non-chromosomal nucleic acid comprising an intact rep!icon such that the vector may be replicated when placed within a permissive cell, for example by a process of transformation
• a vector may replicate in one cell type, such as bacteria, but have limited ability to replicate m another cell, such as mammalian cells.
• Vectors may be viral or non- viral.
• non- viral vectors for delivering nucleic acid include naked DNA; DNA complexed with cationic lipids, alone or in combination with cationic polymers; anionic and cationic liposomes; DNA-protem complexes and particles comprising DNA condensed with cationic polymers such as heterogeneous polylysme, defined-length
• oligopeptides and polyethylene imine, m some cases contained m liposomes; and the use of ternary complexes comprising a virus and polylysine-DNA.
• the term“substantially the same”, used interchangeably with the term “comparable”, or“similar”, when referring to cytotoxicity, viability or cell recovery, refers to the that the tw ? o measurements of cytotoxicity, viability or cell doubling time are no more than 25%, no more than 20%, no more than 15% different, no more than 10%, no more than 8%, or no more than 5% different from each other.
• cytotoxic when used to describe the activity of effector cells such as NK cells, relates to killing of target cells by any of a variety of biological, biochemical, or biophysical mechanisms.
• This disclosure provides a culture medium customized for growing N K.--92 : cells (“the customized NK-92 !3 ⁇ 4 culture medium”), which comprises a basal medium and one or more supplements, the one or more supplements comprising ethanolamine, an ethanolamine derivative, insulin, transferrin, sodium selenite, or a combination thereof.
• a basal medium disclosed herein refer to an unsupplemented cell culture medium. Basal medium typically contains inorganic salts, carbon source, vitamins and amino acids. Suitable basal media include but not limited to Isocove’s Modified Dulbecco’s Medium (IMDM) and Mimimum Essential Medium Eagle alpha modification, Roswell Park Memorial Istitute (RPMI) 1640 Medium, McCoys 5A Modified Medium. Many of these media, e.g , IMDM, can be obtained
• the basal culture medium comprises saccharides.
• Exemplary vitamins that can be used in the basal medium may be one or more vitamins selected from the group consisting of biotin, choline chloride, D-calcium pantothenate, folic acid, niacinamide, pyndoxal hydrochloride, riboflavin, thiamine hydrochloride, vitamin B12, and i- inositol.
• Exemplary inorganic salts that can be used in the basal medium may be one or more inorganic salts selected from the group consisting of CaCk, MgS0 4 , KC1, KNO3, NaHCCb,
• Exemplary amino acids that can he used in the basal medium may be one or more amino acids selected from the group consisting of L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-eystine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L- lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L- tyrosine and L- valine.
• the basal medium may also comprise carbohydrates, such as saccharides including glucose.
• the basal medium comprises about 3-6 g/L glucose.
• the basal medium may also comprise sodium selenite, e.g., about 10-25 ug/L, e.g., 17 ug/L.
• the basal medium may include buffer components such as 4-(2- hydroxyethyi)-l-piperazmeethanesulfonic acid (HEPES).
• the basal medium may include one or more other components such as phenol red, hypoxanthme monosodium salt pyruvates, linoleic acid, lipoic acid, putrescine dihydrochloride, thymidine and so forth.
• the basal medium includes sodium chloride, sodium bicarbonate, sodium phoshage monobasic, potassium chloride, calcium chloride, glucose, and HEPES.
• the basal medium comprises 2-6 g/L sodmm chloride (e.g., 4-5 g/L, or 4.505 g/L sodium chloride), 1-5 g/L sodium bicarbonate (e.g., 2-4 g/L, or 3.024 g/L sodium bicarbonate), 0.1-0.6 g/L potassium chloride (e.g., 0.2-0.4 g/L, or 0.33 g/L potassium chloride), 0.1-0.4 calcium chloride (e.g., 0.15-0.2 0.1653 calcium chloride, 0.05-0.2 g/L sodium phoshage monobasic (e.g., 0.109 g/L sodium phoshage monobasic), 2-8 g/L glucose (e.g., 4.5 g/L glucose), 2-8 g/L HEPES (e.g., 5.958 g/L HEPES).
• sodmm chloride e.g., 4-5 g/L, or 4.505 g/L sodium chlor
• the customized NK-92 ® culture medium disclosed herein comprises the basal medium as described above plus one or more supplements, including but not limited to ethanolamme, an ethanolamme derivative, insulin, transferrin, or a combination thereof.
• Albumin is a protein supplement in cell culture used to deliver unesterified fatty acids into and from cells; albumin can be, for example, human albumin (HA) or bovine serum albumin (BSA).
• the albumin is human albumin or HA.
• Human albumin (HA) is the most copious protein in human serum at approximately 3.5 - 5.0 g/dL and functions as a carrier protein for steroids and fatty acids m blood.
• Human albumin is commercially available, for example, from CSL Behring, King of Prussia, PA, Gnffiois, Octapharma etc.
• the basal medium is supplemented with 0.05-1.0% HA, e.g., 0.1-1.0%, 0.125%, 0.5%, or 1.0% of HA.
• the customized NK-92 ® culture medium may comprise human AB serum.
• Human AB serum is produced by clotting human whole blood and separating the liquid phase from the clotted solid phase.
• human AB serum contains components that are absent in HA, e.g., additional proteins (that are not used in clotting), electrolytes, antibodies, glucose, and hormones.
• the basal medium is supplemented with 1-7%, e.g., 3-7%, or 5% human AB serum.
• Ethanoiamine is an organic chemical compound with the formula HOCH2CH2NH2. The molecule is both a primary amine and a primary alcohol (due to a hydroxyl group). Ethanoiamine is a colorless, viscous liquid and typically used in the production of detergents, emulsifiers, polishes, pharmaceuticals, corrosion inhibitors, and chemical intermediates. Ethanoiamine is also a precursor of phospho-glycerides which are essential to the structure of the plasma membrane and cellular organelles. [0070] Ethanolamine derivatives are compounds that are derived from ethanolamine, e.g., by a chemical reaction, and comprise similar chemical structure.
• the ethanolamine derivatives can be ethanolamides, which are formed by a condensation reaction between ethanolamine and carboxylic acids.
• Ethanolamine derivatives may also be an ethanolamine phospholipid, e.g., phosphatidylethanolamine.
• Non-limiting examples of ethanolamides that can be used include vaccenic acid ethanolamide (VEA) (e.g., cis- vaccenic acid ethanolamide), oleic acid ethanolamide (OEA) (e.g., cis- oleic acid ethanolamide), palmitic acid ethanolamide (PAE), and stearic acid ethanolamide (SEA). These compounds are fatty acid ethanolamides that typically can be found m human and rat blood plasma.
• the basal medium disclosed herein may be supplemented with one or more supplements.
• the one or more supplements comprise 0.2-20 mg/L ethanolamine and/or ethanolamine derivatives, e.g., 5-10 mg/L, 10-20 mg/L, 1-10 mg/L, 1-5 mg/L, or 2 mg/L.
• other supplements may be used including one or more of insulin, transferrin, and selenium.
• Insulin promotes glucose and amino acid uptake, lipogenesis, intracellular transport, and the synthesis of proteins and nucleic acids.
• Transferrin is an iron carrier and may also help to reduce toxic levels of oxygen radicals and peroxide.
• Selenium, as sodium selenite is a co-factor for glutathione peroxidase and other proteins that is used as an anti-oxidant in media the basal medium is supplemented with a solution comprising a mixture of insulin, transferrin, selenium, and ethanolamine.
• the basal medium in the customized NK-92 ® culture medium is supplemented with insulin, transferrin, and sodium selenite at amounts that are suitable for ceil culture.
• the basal medium may be supplemented with 1 -20 mg/L, e.g., 5-10 mg/L, or 10 mg/L insulin; 2.5-12 mg/L, e.g., 5.5 mg/L transferrin, and/or 6.7 ug/L sodium selenite.
• a supplement added to the basal medium to form the NK-92 ® culture medium is a pre-rnade concentrated mixture of multiple components, such as insulin, transferrin, selenium, and ethanolamine and is diluted to a working concentration when it is added to the basal medium.
• the pre-rnade mixture may comprise, for example, 500-10,000 mg/L insulin, 250-10,000 mg/L transferrin, 0.25-15.0 mg/L sodium selenite, and 100-4,000 mg/L ethanolamine, and the pre-made mixture can be added to the basal medium at suitable dilutions, for example, 1 : 100, or 1 : 1000.
• the one or more supplements that are added to the basal medium comprise poloxamer 188 (poloxamer 188 F68).
• the produced customized NK-92 ® culture medium comprises 0.01-0.1%, e.g., 0.05% poloxamer 188.
• the customized NK-92 ® culture medium comprises 4.5-20 g/L, e.g., 10-20 g/L, 5-10 g/L, about 6.5 g/L, about 9 g/L, 11 g/L, 13 g/L, 15 g/L, 17 g/L glucose.
• NK-92 ® cells are aNKTM cells, which does not produce endogenous interleukin-2 (IL-2)
• 1L-2 is also supplemented to the basal medium at an amount sufficient to support the aNKTM ceils growth.
• IL-2 is supplemented to an amount of 300-600 IU/mL e.g., 500 IU/mL in the customized NK-92 ® culture medium.
• the NK-92 ® ceils that have been grown in the customized NK-92 ® culture medium have direct cytotoxicity and/or ADCC that is substantially the same as that of NK-92 ® cells that have been grown in a reference growth medium. Direct cytotoxicity and ADCC at different growth cycles, e.g., cycle 3, cycle 4, and/or cycle 5, may be measured using the methods described below.
• the NK-92 ® cells that have been grown in the customized NK- 92 ® culture medium have direct cytotoxicity' and/or ADCC that is 1-30%, e.g., 1-15%, higher than that of the NK-92 ® cells grown m the reference growth medium.
• the NK-92 ® cells that been growm in the customized NK-92 ® culture medium have substantially the same direct cytotoxicity and/or ADCC as those of the NK-92 ® cells grown in the reference growth medium.
• An illustrative example is shown m Example 2, where Group G shows substantially the same cytotoxicity to the NK-92 ® cells grown in reference culture medium.
• the customized NK-92 ® culture medium comprises a basal medium that is supplemented with ethanolamine but not HA.
• the ethano!amine may be 0.05-40 mg/L, e.g., 0.2- 20 mg/L, or 2 mg/L.
• An illustrative example is shown in Example 2, Table 5, Groups D and F, in which aNKTM cells grown in customized NK-92 ® culture medium show3 ⁇ 4d substantially the same growth rate, viability, and cytotoxicity as the aNKTM cells that have grown in the reference growth medium.
• the basal medium is further supplemented with transferrin, insulin and sodium selenite.
• the basal medium is supplemented with ethanolamme and HA.
• the HA in the customized NK-92 ® culture medium can be 0.05-1.0%, e.g., 0.125-1.0%, 0.25%, or 0.5%, and ethanolamine can be 2.0 mg/L.
• Table 5 e.g., Groups G and I.
• the basal medium is supplemented with ethanolamme, glucose, insulin, transferrin, and sodium selenite.
• ethanolamme e.g., glucose, insulin, transferrin, and sodium selenite.
• Table 5 e.g., Group F.
• the basal medium is supplemented with ethanolamme, HA, and glucose. Ethanolamme and HA may be supplemented in the amounts as described above. Glucose can he added to the basal medium such that the final concentration in the customized NK-92 ® medium is 4.5-20 g/L.
• An illustrative example is shown m Table 7, e.g., Group I.
• the basal medium is further supplemented with insulin, transferrin, and sodium selenite in suitable amounts disclosed above.
• the basal medium comprises 4.5 g/L glucose and is supplemented with an additional amount of 2.0g/L glucose (such that the final concentration in the customized NK-92 ® culture medium is 6.5 g/L), 2.0 mg/L ethanolamine, and 1.0% human albumin.
• the basal medium is supplemented with 0.2-20 mg/L ethanolamine, 0.125-0.5% of HA, and glucose in an amount such that the concentration of glucose in the customized NK-92 ® culture medium is about 4.5-20 g/L, e.g., 6.5 g/L.
• the basal medium comprises 4.5 g/L glucose and is supplemented with an additional amount of 2.0g/L glucose (such that the final concentration m the customized NK-92 ® culture medium is 6.5 g/L), 10 mg/L insulin, 5.5 mg/L transferrin, 6.7 ug/L sodium selenite, 0.125% human albumin, and 2.0 mg/L ethanolamine.
• human AB serum and/or po!oxamer 188 are also added to the basal medium that are supplemented with the various combinations of supplements described herein.
• the customized NK-92 ® culture media comprising the basal media supplemented with the various combinations of the supplements disclosed herein support a growth rate, viability', direct cytotoxicity, and/or ADCC that is substantially the same as the NK-92 ® cells that have been grown in the reference growth medium.
• Other growth promoting substances include,
• Other cell gro wth promoting substances may also he used to supplement the basal medium for culturing NK-92 ® cells.
• These substances include, but not limited to, nucleosides, 2- ketoglutaric acid (2-oxoglutaric acid), fructose, galactose, glycerophosphoric acid, citric acid, ethanolamine, para-aminobenzoie acid, iron- containing compounds, such as FeS0 4 and hemin, benzamidine, putrescine, and unsaturated fatty acids, such as oleic acid and linolic acid.
• antimicrobial agents such as streptomycin, nystatin, gentamicins, ciprofloxacin, norfloxacin and levofloxacin, may be used in combination with the supplements mentioned above.
• NK-92 ® cells typically starts from thawing frozen NK-92 ® cells and seeding them in a container with a suitable medium. Cells are allowed to recover until the cell viability reaches a certain value, for example, greater than 85%. Cells are then expanded in a vessel, e.g., a T-Flask or G-Rex vessel, to a desirable cell density, for example, a density that is equal to or less than l 2xl0 6 cells/mL. The cell culture from the vessel is then collected and used to inoculate one or more larger culture vessels in the customized NK-92 ® culture medium as disclosed above.
• a vessel e.g., a T-Flask or G-Rex vessel
• wTiich can have a volume of at least 2 liters, at least 5 liters, at least 10 liters, or at least 25 liters. Transfer of cells between different vessels can be performed using means well known in the art, e.g., using a serological pipette, pump or a gravity feed, performed under sterile conditions.
• NK-92 ® cells so produced can be harvested by centrifugation.
• the centrifugation is performed using a continuous centrifuge aseptical!y attached to the culture vessel, e.g., the single use culture bags for a wave bioreactor, at the end of the expansion process.
• the culture supernatant is then removed and the cells are resuspended in a wash buffer.
• the wash can be repeated for at least two times, at least three times, e.g., 4-6 times.
• the mixture containing the cells and wash buffer can be centrifuged again and the cells are collected and processed for therapeutic applications.
• the NK-92 ® cells grown in the customized NK-92 ® culture medium as described above are assessed for cytotoxicity.
• Direct cytotoxicity of the produced NK-92 ® cells can be assessed by methods well known in the art, for example, a 5l Cr release assay (Gong et al., Leukemia, Apr; 8(4): 652-658 (1994)) using the procedure described by Klingemann et al.
• direct cytotoxicity of the produced NK-92 ® cells can be assessed using a calcein release assay.
• the NK-92 ® ceils (referred to as the effector in the assay) can be mixed with the calcein loaded target cells (referred to as target in the assay) at certain ratios.
• the calcein released from the target cells can be assessed, e.g., by a fluorescence plate reader.
• the ratio of the effector and target used m the assay may vary, optionally the effector: target ratio may be 20: 1, 15: 1, 10: 1, 8: 1, 5: 1, 2.5: 1, 1.25: 1, 0.625: 1, 0.31 : 1, 0.16: 1, 0.08: 1, 0.04: 1, 0.02: 1 ; optionally the effector: target ratio is 10: 1.
• the target cells can be any cells that express MHC molecules that can be recognized by the NK-92 ® cells, for example, K562 cells, or BT-474 cells.
• the values of cytotoxicity of NK-92 ® cells may vary depending on the type of target cells used as well as the effectortarget ratio and the growth cycle which the NK-92 ® cells are in.
• the NK-92 ® cells produced using the methods described herein can have a direct cytotoxicity' of at least 50-100%, e.g., 60-100%, 70-100%, or 80-100%.
• aNKTM cells or haNK ® cells may have a direct cytotoxicity' of 80- 110% when using K562 cells as the target cells, e.g., 82-100%, 85-100%, 87-100%, 88-100%, or 89-100%, by a calcein release assay, when the NK ⁇ 92 CE cells are at a growth cycle selected from growth cycles 3-12, e.g., cycle 3, 4, or 5.
• the cytotoxicity of NK-92 ® cells is the antibody dependent cellular cytotoxicity (ADCC).
• ADCC antibody dependent cellular cytotoxicity
• Methods for measuring the ADCC of NK- 92 ® cells are similar to the methods of measuring direct cytotoxicity as described above except that an antibody that can recognize the target cell is added.
• the Fe receptor of the NK ceils recognizes the cell-bound antibodies and triggers cytolytic reaction and killing the target cells.
• the haNK ® cells can be incubated with Ramos (target cells) in the presence of Rituxan (anti-CD20 antibody) and killing of the Ramos cells can be measured by the release of internal components of the target cells, e.g., 5l Cr or calcein, as described above.
• the ratio of the effector and target used in the assay may vary, optionally the effector: target ratio may be 20: 1, 15: 1, 10: 1, 8: 1, 5: 1, 2.5: 1, 1.25: 1 , 0.625: 1, 0.31 : 1, 0.16: 1, 0.08: 1,0.04: 1, or 0.02: 1; preferably the effector: target ratio is 10: 1.
• haNK ® cells have a ADCC toxicity of at least 60%, at least 70%, at least 80%, or at least 90% when tested at an effector: target ratio of 10: 1.
• haNK ® cells may have an ADCC cytotoxicity of 60-120%, e.g., 80-120%, 90- 115%, 97-110%, or 100-120% when the haNK ® cells are at a growth cycle selected from growth cycles 3-12, e.g., cycle 3, 4, or 5, when using an effector: target ratio of 10: 1.
• the NK-92 ® ceils grown in the customized NK-92 ® culture medium may have substantially the same direct cytotoxicity and/or ADCC as those of the NK-92 ® cells grown in the reference growth medium.
• the NK-92 ® cells grown in the customized NK-92 ® cells have a direct cytotoxicity and/or ADCC that is at 90-120%, of that of the NK-92 ® ceils that have been grown in the reference growth medium.
• the growth rate of NK-92 ® cells can be assessed using cell doubling time, i.e., the time it takes for the cells to proliferate and reach twice the initial cell number.
• the doubling time is inversely related to the growth rate of the NK-92 ® cells: the greater the doubling time, the lower the growth rate.
• the NK-92 ® cells that have been grown in the customized NK-92 ® culture medium have a doubling time of 30-50 hours, e.g., 30-40, 40-50, or 40-47 hours.
• Methods for measuring cell viability are also well known, for example, trypan blue exclusion assay, in which dead cells are stained blue and viable cell number can be calculated by substracting the trypan blue stained cells from the total cells.
• Cell counting can be performed on a counting chamber of a hemocytometer.
• Automatic cell counting based on the fact that cells show great electrical resistance, are also commonly used to count cells as well as measure their volume.
• the automatic cell counter is the Coulter counter, available from Beckman Coulter, Brea, CA.
• Another example is the NC-200TM Nueleocounter automated cell counter, available from Chemometec, Denmark. This device enumerates live and dead cells based on their staining by fluorescent viability dyes.
• the NK-92 ® cells that have grown in the customized NK-92 ® culture medium may have 85-100%, viability.
• the cytotoxicity, viability, and/or growth rate of the NK-92 ® cells grown in the media disclosed herein are compared with NK-92 ® cells that have been grown in a reference growth medium.
• the reference growth medium can be any medium that one of ordinary skill in the art have used to culture NK-92 ® cells in some embodiments, the reference growth medium may comprise cytokines that are necessary for growth of certain NK-92 ® cells, for example, IL- 2.
• the reference growth medium is further supplemented wath human AB serum, poloxamer 188 if the customized NK-92 ® culture medium is also supplemented with these ingredients.
• IL-2 is also supplemented in the reference growth medium.
• the reference growth medium also comprises L-Serine (e.g., at a concentration of 0.324 mmol/L), L- Asparagine (e.g., at a concentration of 0.036 mmol/L), L-Glutamine (e.g., at a concentration of 0.45 mmol/L).
• the reference growth medium comprises human AB serum, IL-2, and poloxamer 188.
• the NK-92 ® ceils that can be cultured using the methods disclosed herein include aNKTM cells, haNK ® cells, taNK ® and t- haNK ® (e.g., CD19 t-haNK ® , PD-L1 t-haNK ® , or HER.2-t-haNK ® ) cells, which are further described below ' .
• the NK-92 ® cell line is a unique cell line that was discovered to proliferate in the presence of interleukin 2 (IL-2). Gong et ai., Leukemia 8:652-658 (1994). These cells have high cytolytic activity against a variety' of cancers.
• the NK-92 ® cell line is a homogeneous cancerous NK cell population having broad anti-tumor cytotoxicity with predictable yield after expansion. Phase I clinical trials have confirmed its safety' profile.
• NK-92 ® was discovered in the blood of a subject suffering from a non-Hodgkins lymphoma and then immortalized ex vivo.
• NK-92 ® cells are derived from NK cells, but lack the major inhibitory' receptors that are displayed by normal NK cells, while retaining the majority of the activating receptors. NK-92 ® cells do not, however, attack normal cells nor do they elicit an unacceptable immune rejection response in humans. Characterization of the NK-92 ® cell line is disclosed in WO 1998/49268 and U.S. Patent Application Publication No. 2002-0068044.
• the NK-92 ® cell line is found to exhibit the CD56 bright , ( 1)2. CD7, CD 11 a, CD28, CD45, and CD 54 surface markers. It furthermore does not display the CD1 , CD 3, CD4, CDS, CDS, CD 10, (4) 1 1. CD 16, (4) 19. CD20, ( 1)23. and CD34 markers. Growth of NK-92 ® cells in culture is dependent upon the presence of recombinant interleukin 2 (rIL-2), with a dose as low as 1 lU/mL being sufficient to maintain proliferation. NK-92 ® has high cytotoxicity even at a low effector: target (E:T) ratio of 1 : 1. Gong, et a!., supra. NK-92 ® cells are deposited with the American Type Culture Collection (ATCC), designation CRL-2407.
• ATCC American Type Culture Collection
• Modified NK-92 ® cells are known and include, but are not limited to, those described m, e.g., U.S. Patent Nos. 7,618,817, 8,034,332, and 8,313,943, U.S. Patent Application
• NK-92 ® cells retain almost all of the activating receptors and cytolytic pathways associated with NK cells, they do not express CD 16 on their cell surfaces.
• CD 16 is an Fc receptor which recognizes and binds to the Fc portion of an antibody to activate NK ceils for antibody-dependent cellular cytotoxicity (ADCC). Due to the absence of CD 16 receptors, NK- 92 ® cells are unable to lyse target cells via the ADCC mechanism and, as such, cannot potentiate the anti-tumor effects of endogenous or exogenous antibodies (i.e., rituxan and herceptin).
• ADCC antibody-dependent cellular cytotoxicity
• IL-2 1000 IU/mL
• IL-2 1000 IU/mL
• NK-92 ® is a cancer- derived cell line
• endogenous NK cells are harvested from a donor (or the patient) and processed for infusion into a patient.
• Endogenous NK cell preparations are heterogeneous cell populations, whereas NK-92 ® cells are a homogeneous, clonal cell line. NK- 92 ® cells readily proliferate in culture while maintaining cytotoxicity, whereas endogenous NK cells do not. In addition, an endogenous heterogeneous population of NK cells does not aggregate at high density. Furthermore, endogenous NK cells express Fc receptors, including CD- 16 receptors that are not expressed by NK-92 ® cells.
• Fc receptors bind to the Fc portion of antibodies.
• Fc receptors are known, and differ according to their preferred ligand, affinity, expression, and effect following binding to the antibody.
• NK-92 ® cells are modified to express an Fc receptor protein on the cell surface.
• the Fc receptor is CD 16.
• a representative amino acid sequence encoding CD16 is shown in SEQ ID NO:2.
• a representative polynucleotide sequence encoding CD16 is shown in SEQ ID NQ: i .
• NK-92 ® cells are modified by introducing a polynucleotide encoding a CD16 polypeptide has at least about 70% polynucleotide sequence identity with a polynucleotide sequence encoding a full-length, including signal peptide, naturally occurring CD16 that has a phenylalanine at position 176 of the full-length CD16.
• a polynucleotide encoding a CD16 polypeptide has at least about 70%
• polynucleotide sequence identity with a polynucleotide sequence encoding a full-length, including the signal peptide, naturally occurring CD 16 that has a valine at position 176.
• Homologous polynucleotide sequences include those that encode polypeptide sequences coding for variants of CD 16.
• homologous CD 16 polynucleotides may be about 150 to about 700, about 750, or about 800 polynucleotides in length, although CD16 variants having more than 700 to 800 polynucleotides are within the scope of the disclosure.
• cDNA sequences having polymorphisms that change the CD16 ammo acid sequences are used to modify the NK-92 ® cells, such as, for example, the allelic variations among individuals that exhibit genetic polymorphisms in CD 16 genes.
• CD 16 genes from other species that have a polynucleotide sequence that differs from the sequence of human CD 16 are used to modify NK-92 ® cells.
• variant polypeptides are made using methods known in the art such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis.
• Site direct mutagenesis Carter, 1986; Zoller and Smith, 1987
• cassette mutagenesis restriction selection mutagenesis
• Wells et al , 1985 or other known techniques can be performed on the cloned DNA to produce CD 16 variants (Ausubei, 2002; Sambrook and Russell, 2001).
• CD16 polypeptide variants are at least 200 amino acids m length and have at least 70 % ammo acid sequence identity, or at least 80%, or at least 90% identity to SEQ ID NO: 1 or SEQ ID NQ:2. In some embodiments, CD16 polypeptide variants are at least 225 amino acid in length and have at least 70 % amino acid sequence identity , or at least 80%, or at least 90% identity to SEQ ID NO: 1 or SEQ ID NO: 2.
• a nucleic acid encoding a CD 16 polypeptide may encode a CD 16 fusion protein.
• a CD 16 fusion polypeptide includes any portion of CD 16 or an entire CD 16 fused with a non-CD 16 polypeptide.
• a fusion polypeptide may be created in which a heterologous polypeptide sequence is fused to the C-terminus of CD 16 or is positioned internally in the CD 16. Typically, up to about 30 % of the CD 16 cytoplasmic domain may be replaced. Such modification can enhance expression or enhance cytotoxicity (e.g.,
• chimeric proteins such as domains from other lymphocyte activating receptors, including but not limited to Ig-a, Ig-B, CD3-e, CD3-d, DAI 3 - 12 and DAP- 10, replace a portion of the CD 16 cytoplasmic domain.
• Fusion genes can be synthesized by conventional techniques, including automated DNA synthesizers and PCR amplification using anchor primers that give rise to complementary' overhangs between two consecutive gene fragments that can subsequently be anneal ed and re- amplified to generate a chimeric gene sequence (Ausubel, 2002). Many vectors are commercially available that facilitate sub-cloning CD 16 in-frame to a fusion moiety.
• NK-92 ® cells are further engineered to express a chimeric antigen receptor (CAR) on the cell surface.
• CAR chimeric antigen receptor
• the CAR is specific for a tumor- specific antigen.
• Tumor-specific antigens are described, hy way of non-limiting example, in U.S. 2013/0189268; WO 1999024566 Al ; U.S. 7,098,008; and WO 2000020460 Al, each of which is incorporated herein by reference in its entirety.
• Tumor-specific antigens include, without limitation, NKG2D, CSi, GD2, CD 138, EpCAM, EBNA3C, GPA7, CD244, CA-125, ETA, MAGE, CAGE, BAGE, HAGE, LAGE, PAGE, NY-SEQ-l, GAGE, CEA, CD52, CD30, MUC5AC, c-Met, EGER, FAB, WT-1, PSMA, NY-ESOl, AFP, CEA, C l AG! B. CDI9 and CD33. Additional non-limiting tumor-associated antigens, and the malignancies associated therewith, can be found in Table 2. Table 2: Tumor-Specific Antigens and Associated Malignancies
• the CAR targets GDI 9, CD33 or CSPG-4.
• variant polypeptides are made using methods known in the art such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis.
• Site direct mutagenesis Carter, 1986; Zol!er and Smith, 1987
• cassette mutagenesis restriction selection mutagenesis
• Wells et al , 1985 or other known techniques can be performed on the cloned DNA to produce CD 16 variants (Ausubel, 2002; Samhrook and Russell, 2001)
• the CAR targets an antigen associated with a specific cancer type.
• the cancer is selected from the group consisting of leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including
• myeloblastic promyelocytic, myelomonocytic, monocytic, and erythroleukemia)
• chronic leukemias e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia
• polycythemia vera lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulmemia, heavy chain disease, solid tumors including, but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,
• lymphangiosarcoma iymphangioendotheliosareoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wiim's tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, epend
• a polynucleotide encoding a CAR is mutated to alter the amino acid sequence encoding for CAR without altering the function of the CAR.
• polynucleotide substitutions leading to amino acid substitutions at“non-essential” ammo acid residues can be made in the CARs disclosed above.
• CARs can be engineered as described, for example, in Patent Publication Nos. WO 2014039523; US 20140242701; US 20140274909; US 20130280285; and WO 2014099671, each of which is incorporated herein by reference in its entirety.
• the CAR is a CD19 CAR, a CD33 CAR or CSPG-4 CAR.
• cytotoxicity of NK-92 ® cells is dependent on the presence of cytokines (e.g., interleukin-2 (IL-2).
• IL-2 interleukin-2
• the cost of using exogenously added IL-2 needed to maintain and expand NK-92 ® cells in commercial scale culture is significant.
• the administration of IL-2 to human subjects in sufficient quantity to continue activation of NK-92 ® cells would cause adverse side effects.
• FcR-expressing NK-92 ® cells are further modified to express at least one cytokine and a suicide gene.
• the at least one cytokine is IL-2, IL-12, IL-15, IL-18, IL-21 or a variant thereof.
• the cytokine is IL-2.
• a representative nucleic acid encoding IL-2 is shown m SEQ ID NO: 3 and a representative polypeptide of IL-2 is shown in SEQ ID NO:4.
• the IL-2 is a variant that is targeted to the endoplasmic reticulum.
• the IL-2 is expressed with a signal sequence that directs the IL-2 to the endoplasmic reticulum. Not to be bound by theory, but directing the IL-2 to the endoplasmic reticulum permits expression of IL-2 at levels sufficient for autocrine activation, but without releasing IL-2 extrace!!ulariy. See Konstantimdis et al“Targeting IL-2 to the endoplasmic reticulum confines autocrine growth stimulation to NK-92 ® cells” Exp. Hemato!. 2005
• suicide gene is one that allows for the negative selection of the cells.
• a suicide gene is used as a safety system, allowing the cells expressing the gene to be killed by introduction of a selective agent. This is desirable m case the recombinant gene causes a mutation leading to uncontrolled cell growth.
• a number of suicide gene systems have been identified, including the herpes simplex virus thymidine kinase (TK) gene, the cytosine deaminase gene, the varicella- zoster virus thymidine kinase gene, the nitroreductase gene, the Escherichia coli gpt gene, and the E.
• the suicide gene is active in NK-92 ® cells.
• the suicide gene encodes for a protein that has no ill-effect on the cell but, in the presence of a specific compound, will kill the cell.
• the suicide gene is typically part of a system.
• the suicide gene is the thymidine kinase (TK) gene.
• the TK gene may be a wild-type or mutant TK gene (e.g ., tk30, tk75, sr39tk). Cells expressing the TK protein can be killed using ganciclovir.
• the suicide gene is Cytosine deaminase which is toxic to cells in the presence of 5-fluorocytosine.
• the suicide gene is cytochrome P450 which is toxic in the presence of ifosfamide, or cyclophosphamide. See e.g.,Touati et al.“A suicide gene therapy combining the improvement of cyclophosphamide tumor cytotoxicity and the development of an anti-tumor immune response.” Curr Gene Ther. 2014; 14(3):236-46.
• the suicide gene is iCas9 Di Stasi, (2011)“Inducible apoptosis as a safety switch for adoptive cell therapy.” N Engl J Med 365: 1673-1683. See also Morgan, “Live and Let Die: A New Suicide Gene Therapy Moves to the Clinic” Molecular Therapy (2012); 20: 1 1-13.
• the iCas9 protein induces apoptosis in the presence of a small molecule API 903.
• API 903 is biologically inert small molecule, that has been shown in clinical studies to be well tolerated, and has been used in the context of adoptive cell therapy.
• the modified NK-92 ® cells are irradiated prior to administration to the patient. Irradiation of ⁇ K -02 cells is described, for example, in U.S. Patent No. 8,034,332, which is incorporated herein by reference in its entirety. In one embodiment, modified NK-92 & cells that have not been engineered to express a suicide gene are irradiated.
• Transgenes e.g., CD 19. CAR and CD16
• Transgenes can be engineered into an expression vector by any mechanism known to those of skill in the art.
• Transgenes may be engineered into the same expression vector or a different expression vector.
• the transgenes are engineered into the same vector.
• the vector allows incorporation of the transgenefs) into the genome of the cell.
• the vectors have a positive selection marker.
• Positive selection markers include any genes that allow the cell to grow under conditions that would kill a cell not expressing the gene.
• Non-limiting examples include antibiotic resistance, e.g., geneticin (Neo gene from Tn5).
• the vector is a plasmid.
• the vector is a viral vector.
• Viral vectors include, but are not limited to, retroviral vectors, adenoviral vectors, adeno-associated viral vectors, herpes simplex viral vectors, pox viral vectors, and others.
• Transgenes can be introduced into the NK-92 ® cells using any transfection method known in the art, including, by way of non-limiting example, infection, electroporation, lipofection, nucleofection, or“gene- gun.”
• any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of this disclosure including, but not limited to, steps in methods using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed, it is understood that each of these additional steps can be performed with any specific method steps or combination of method steps of the disclosed methods, and that each such combination or subset of combinations is specifically contemplated and should be considered disclosed.
• compositions disclosed herein include the following exemplary embodiments.
• Embodiment 1 A method of culturing NK-92 ® cells comprising culturing NK-92 ® cells in a customized NK-92 ® culture medium comprising a basal medium and one or more supplements, wherein the one or more supplements comprise ethanolamine, an ethanolamine derivative, insulin, transferrin, sodium selenite, HA, or a combination thereof; and wherein the basal medium comprises inorganic salts, vitamins and amino acids.
• Embodiment 2 The method of embodiment 1, wherein the ethanolamine derivative is an ethanolamide
• Embodiment 3 The method of embodiment 2, wherein the ethanolamide is vaccenic acid ethanolamide, or oleic acid ethanolamide, palmitic acid ethanolamide, or stearic acid ethanolamide.
• Embodiment 4 The method of embodiment 1 , wherein the ethanolamine derivative is phosphatidylethanolamine.
• Embodiment 5 The method of any of embodiments 1-4, wherein the one or more supplements comprise 1-7% human AB serum.
• Embodiment 6 The method of any of embodiments 1-5, wherein the one or more supplements further comprise insulin, transferrin, selenium, or a combination thereof.
• Embodiment 7 The method of any of embodiments 1-6, wherein the one or more supplements further comprise 300-600 RT/mL interleukin-2.
• Embodiment 8 The method of any of embodiments 1-7, wherein the one or more supplements further comprise 0.01% to 0.1% of poloxamer 188.
• Embodiment 9 The method of any of embodiments 1-8, wherein the NK-92 ® cells cultured m the customized NK-92 ® culture medium have substantially the same or higher cytotoxicity, growth rate, and viability compared to NK-92 ® cells grown in a reference growth medium.
• Embodiment 10 The method of any of embodiments 1-9, wherein the NK-92 ® cells cultured in the customized NK-92 ® culture medium have 85-100% viability.
• Embodiment 11 The method of any of embodiments 1-10, wirerein the NK-92 ® cells cultured in the customized NK-92 ® culture medium show a direct cytotoxicity of and/or an ADCC of 60-100% at an effector to target ratio of 10: 1.
• Embodiment 12 The method of any of embodiments 1-11, wherein the NK-92 ® cells cultured in the customized NK-92 ® culture medium have a doubling time of 30-50 hours.
• Embodiment 13 The method of any of embodiments 1-12, wherein the NK-92 ® cells express a cytokine, Fc Receptor, a chimeric antigen receptor, or a combination thereof.
• Embodiment 14 The method of embodiments 1-13, wherein the customized NK- 92 ® culture medium comprises 0.05-40 mg/'L of ethanoJamine, an ethanoJamine derivative, or a combination thereof.
• Embodiment 15 The method of embodiments 1-14, wherein the customized NK- 92 ® culture medium 4.5-20 g/L of glucose.
• Embodiment 16 The method of any of embodiments 1-15, wherein the customized NK-92 ® culture medium is further supplemented with 0.05% to 1.0% HA.
• Embodiment 17 A ceil culture comprising NK-92 ® ceils in a customized NK-92 ® culture medium composing a basal medium and one or more supplements, wherein the one or more supplements comprise ethano!amine, an ethano!amine derivative, insulin, transferrin, sodium selenite, HA, or a combination thereof; and wherein the basal medium comprises inorganic salts, vitamins and amino acids.
• Embodiment 18 A cell culture comprising NK-92 ® cells in a customized NK-92 ® culture medium comprising a basal medium and one or more supplements, wherein the one or more supplements comprise ethanolamine, an ethanolamine derivative, or a combination thereof; and wherein the basal medium comprises inorganic salts, vitamins and amino acids.
• Embodiment 19 The cell culture of embodiment 17 or 18, wherein the
• ethanolamine derivative is ethanolamide.
• Embodiment 20 The cell culture of embodiment 19, wherein the ethanolamide is cis-vaccenic acid ethanolamide or oleic acid ethanolamide.
• Embodiment 21 The cell culture of embodiment 18, wherein one or more supplements further comprise insulin, transferrin, selenium, or a combination thereof.
• Embodiment 22 The cell culture of any of embodiments 17-21, wherein the one or more supplements further comprise 0.01% to 0.1% of poloxamer 188.
• Embodiment 23 The cell culture of any of embodiments 17-22, wherein the customized NK-92 ® culture medium comprises 4.5-20 g/L glucose.
• Embodiment 24 The cell culture of any of embodiments 17-23, wherein the one or more supplements comprise 0.05% to 1.0% HA.
• Embodiment 25 The cell culture of any of embodiments 17-24, wherein the NK- 92 ® cells express a cytokine, Fc Receptor, a chimeric antigen receptor, or a combination thereof.
• Embodiment 26 The cell culture of any of embodiments 17-25, wherein the customized NK-92 ® culture medium comprises insulin, transferrin, selenium, or a combination thereof.
• Embodiment 27 The ceil culture of any of embodiments 17-26, wherein the NK- 92 ® cells have substantially the same or higher cytotoxicity, growth rate, and/or viability as compared to control NK-92 ® cells.
• Embodiment 28 The cell culture of any of embodiments 17-27, wherein the NK- 92 ® cells that have been cultured in the customized NK-92 ® culture medium have 85-100% viability.
• Embodiment 29 The ceil culture of any of embodiments 17-28, wherein the NK- 92 ® cells that have been cultured in the customized NK-92 ® culture medium the doubling time of the NK-92 ® cells is 30-50 hours.
• Embodiment 30 The cell culture of any of embodiments 17-29, wherein the NK- 92 ® cells show a direct cytotoxicity and/or an ADCC of 60-100% when using an effector: target ratio of 10: 1.
• Embodiment 31 The method of any of the embodiments 1-16, wherein the volume of the customized NK-92 ® culture medium is at least 5 titers.
• Embodiment 32 The ceil culture of any of the embodiments 17-30, wherein the cell culture volume is at least 5 titers.
• Embodiment 33 The ceil culture of any of the embodiments 17-30, wherein the NK-92 ® cells maintain substantially the same viability and/or cytotoxicity after the cells are crypreserved and thawed.
• Example 1 Effect of ethanolamine and HA supplementation on aNKTM cells
• aNKTM cells Growth of aNKTM cells in 1MDM based medium (a basal medium) containing different supplements was compared with aNKTM cells growm in a reference growth medium.
• the interleukin-2 (IL-2)-dependent NK-92 ® parental cell line (aNKTM) is presently cultured in a reference growth medium supplemented with human AB serum and IL-2.
• Iscove’s Modified Dulbeco’s Medium (IMDM) supplemented with human AB serum and IL-2 and additional supplements can support the growth, viability and funetionreal activity of the aNKTM cell line in a similar manner to the reference growth medium.
• IMDM Modified Dulbeco’s Medium
• the growth, viability and function of the aNKTM cell line cultured in IMDM basal media containing human AB serum and IL-2 and additional supplements was compared with the reference growth medium.
• Table 3 show3 ⁇ 4 the formulations of various media used in this study.
• Sodium Selenite is present in the IMDM basal media formulation at 17 pg/L.
• the media was supplemented with 6.7 pg/L Sodium Selenite to give a final concentration of 23.7 pg/L Sodium Selenite.
• Glucose is present in the IMDM basal media formulation at 4.5 g/L.
• the media was supplemented with 2 g/L glucose to give a final concentration of 6.5 g/L glucose.
• Sodium selenite was present in the IMDM basal media formulation at 17 pg/L.
• the media was supplemented with 6.7 pg/L sodium selenite to give a final concentration of 23.7 pg/L sodium selenite.
• Glucose was present in the IMDM basal media formulation at 4.5 g/L.
• the media was supplemented with 2 g/L glucose to give a final concentration of 6.5 g/L glucose.
• Average ⁇ standard deviation doubling time was calculated for five growth cycles from viable cell density (VCD) of the aNKTM cell culture determined at the start and end of each growth cycle using an NC200 automated cell counter using the following equation: Ln2/(Ln (VCD End of Cycle/VCD Start of Cyclej/Length of Cycle)).
• Average ⁇ standard deviation viability was calculated for five growth cycles from the cell viability of the aNKTM cell cultures determined at the start and end of each growth cycle using an NC-200 automated cell counter.
• ail basal media were supplemented with human AB serum and interleukin-2 in addition to the supplements shown in the table.
• the average ⁇ standard deviation doubling time was calculated for five growth cycles from viable cell density (VCD) of the aNKTM cell culture determined at the start and end of each growth cycle using an NC-200 automated cell counter using the following equation: Ln2/(Ln (VCD End of Cycle/ VCD Start of Cyeie)/Length of Cycle)).
• Average ⁇ standard deviation viability w3 ⁇ 4s calculated for five growth cycles from the cell viability of the aNKTM cell cultures determined at the start and end of each growth cycle using an NC-200 automated cell counter. Cytotoxicity against K562 cells was determined using a Calcein release assay following standard methods.
• aNKTM cell line cultured in this optimal IMDM formulation was superior to that of aNKTM reference growth medium.
• NK-92 ® reference growth medium or IMDM supplemented with various components were used to grow haNK ® cells.
• the average doubling time, average viability, direct cytotoxicity, and antibody dependent cellular cytotoxicity (ADCC) at the third growth cycle (cycle 3) and at the fifth growth cycle (cycle 5) were assessed and shown in Table 5.
• Groups C and F having identical media compositions except for the presence of insulin, transferrin, selenium, in Group C showed good, and substantially the same, growth rate, viability, direct cytotoxicity and ADCC.
• Group D which has media compositions identical to Group F but for the absence of ethanolamine, had a cell growth rate of 16% slower than that of Group F (Group D showed a cell doubling time of 51 hours as compared to a doubling time of 44 hours in Group F). This indicates that ethanolamine is useful for maintaining desired growth rate.
• Group E having a media composition that is identical to that of Group F but for the absence of HA, showed poor cytotoxicity, only 55% as compared to 90% at cycle 3, and only 57% as compared to 77% at cycle 5, indicating including HA in the customized NK-92 ® culture medium can boost cytotoxicity of the NK-92 ® cells.
• Example 3 Effect of HA titration and other supplements: insulin transferrin and sodium selenite (haNK ® cell study )
• haNK ® cells in IMDM containing different supplements were compared with haNK ® cells in reference growth medium.
• HaNK ® cells (NK-92 ® cells engineered to express GDI 6 and IL-2) is cultured in a reference growth medium comprising human AB serum and poloxamer 188.
• a reference growth medium comprising human AB serum and poloxamer 188.
• Iscove’s Modified Dulbecos Medium (IMDM) supplemented with human AB serum, poloxamer 188 and additional supplements can support the growth, viability and functional activity of haNK ® cells in a similar manner to haNK ® reference growth medium.
• IMDM Modified Dulbecos Medium
• haNK ® cell line cultured in IMDM basal media containing human AB serum, poloxamer 188 and additional supplements was compared with haNK ® reference growth medium. Since a key objective of this experiment was to determine the role of human albumin in supporting cell function, the concentration of this reagent was sequentially increased from 0.125% to 1.0% in groups C to F.
• haNK ® cells were cultured m each media formulation for five growth cycles of 3 to 4 days in T75 tissue culture flasks. For each growth cycle, the cells were grown from -0.3 x lO 6 cells/mL to -0.8-1.2 x 10 6 cells/mL per growth cycle.
• ADCC antibody-dependent cellular cytotoxicity
• Sodium Selenite is present in the IMDM basal media formulation at 17 pg/L The media was supplemented with 6.7 ug/L Sodium Selenite to give a final concentration of 23.7 pg/L Sodium Selenite.
• Glucose is present in the IMDM basal media formulation at 4.5 g/L.
• the media was supplemented with 2 g/L glucose to give a final concentration of 6.5 g/L glucose.
• the media was supplemented with 6.7 pg/L sodium selenite to give a final concentration of 23.7 pg/L sodium selenite.
• Glucose is present in the IMDM basal media formulation at 4.5 g/L.
• the media was supplemented with 2 g/L glucose to give a final concentration of 6.5 g/L glucose.
• Average ⁇ standard deviation doubling time was calculated for five growth cycles from viable cell density (VCD) of the aNKTM cell culture determined at the start and end of each growth cycle using an NC200 automated cell counter using the following equation: Ln2/(Ln (VCD End of Cycle/VCD Start of Cyclej/Length of Cycle)).
• Average ⁇ standard deviation viability was calculated for five growth cycles from the cell viability of the aNKTM cell cultures determined at the start and end of each growth cycle using an NC200 automated cell counter.
• ADCC against Ramos cells and direct cytotoxicity against K562 cells were determined using Calcein AM release assays following standard methods.
• haNK ® ceils exhibited a similar growth rate, viability and functional activity when grown in IMDM basal media containing 0.125% human albumin (Group C versus Group A) with haNK ® cells grown in a reference growth medium.
• human albumin enhanced the functional activity of haNK ® cells
• the growth rate of the ceil line decreased and w r as more variable (Groups D - G) with higher human albumin concentrations.
• absence of human albumin (Group B) resulted in acceptable growth rate, but marginally inferior functional activity, indicating the need for constitui on of this reagent.
• Example 4 Identifying optimal concentrations of human albumin and ethanolamme
• haNK ® cells NK-92 ® engineered to express CD 16 and IL-2
• the medium do not contain insulin, transferrin, or sodium selenite.
• haNK ® cells cultured in an IMDM-basal medium formulation supplemented with human AB serum, poloxamer 188 , 2.0 g/L glucose and a range of concentrations of ethanolamine and human albumin was tested m a multi -parameter format within a 24-well G-Rex plate (Table 8).
• Control groups included in the study included duplicate wells of the reference growth medium and IMDM-basal medium containing human AB serum and poloxamer 188 additionally supplemented with 10 mg/L insulin, 5.5 mg/L transferrin, 6.7 pg/L sodium selenite, 2.0 mg/L ethanolamine, 2.0 g/L glucose, 1.0% human albumin.
• the haNK ® cell line was cultured in each medium formulation for five growth cycles of 3 to 4 days in the G-Rex plate. For each growth cycle, the cells were grown from -0.3 x l 0 b cells/mL to -0.8- 1.2 x 10 6 cells/mL per growth cycle. Cell growth rate and viability was determined by automated cell counting using NC-200TM.
• ADCC antibody-dependent cell cytotoxicity
• wells from columns 1 - 5 contained IMDM-basal medium supplemented with human AB serum, poioxamer 188, 2.0 g/L glucose and the specified concentrations of ethanolamine (EA) and human albumin (HA).
• Control wells A6 and B6 contained reference growth medium;
• Control wells C6 and D6 contained IMDM-basal medium supplemented with human AB serum, poioxamer 188, 10 mg/L insulin, 5.5 mg/L transferrin, 6.7 iig/L sodium selenite, 2.0 mg/L ethanolamine, 2.0 g/L glucose, and 1.0% human albumin.
• Average ⁇ standard deviation doubling time was calculated for five growth cycles from viable cell density (VCD) of the haNK ® ceil culture dete rmined at the start and end of each growth cycle using an automated cell counter using the following equation: Ln2/(Ln (VCD End of Cycle/VCD Start of Cycle)/Length of Cycle)). The doubling time is shown in the top of each cell in the table.
• VCD viable cell density
• Average ⁇ standard deviation viability was calculated for five growth cycles from the cell viability of the haNK ® cell cultures determined at the start and end of each growth cycle using an automated cell counter. The viability is shown in the bottom of each cell in the Table.
• Wells from columns 1 - 5 contained IMDM-basal media supplemented with Human AB Seram, Poloxamer 188, 2 g/L Glsicose and the specified concentrations of Ethanolamine (EA) and Human Albumin (HA).
• EA Ethanolamine
• HA Human Albumin
• Control wells A6 and B6 contained haNK ® reference growth medium; Control wells C6 and D6 contained IMDM-basal media supplemented with Human AB Serum, Poloxamer 188, 10 mg/L Insulin, 5.5 mg/L Transferrin, 6.7 pg/L Sodium Selenite, 2.0 mg/L Ethanolamine, 2 0g/L Glucose, and 1.0% Human Albumin.
• Wells from columns 1 - 5 contained IMDM-basal medium supplemented with human AB serum, poloxamer 188, 2 g/L glucose and the specified concentrations of ethanolamine (EA) and human albumin (HA).
• Control wells A6 and B6 contained reference growth medium;
• Control wells C6 and D6 contained IMDM-basal medium supplemented with human AB serum, poloxamer 188, 10 mg/L insulin, 5.5 mg/L transferrin, 6.7 pg/L sodium selenite, 2 0 mg/L ethanolamine, 2.0 g/L glucose, and 1.0% human albumin.
• test formulations showed superior growth rate or viability to the reference growth medium control, indicating that supplementation with combinations of ethanolamine and human albumin does not compensate for the absence of insulin, transferrin and sodium selenite supplementation in the IMDM-basal formulation.
• the IMDM control formulation exhibited longer doubling times than the reference growth medium control and test formulations, which is consistent wath previous observations that a higher concentration of human albumin inhibits haNK ® ceil growth.
• 1MDM basal media were supplemented with Human AB Seram, Poloxamer 188, 2 g/L Glucose in addition to listed supplements.
• ADCC against Ramos cells and direct cytotoxicity against K562 cells were determined using Caleein AM release assays following standard methods.
• haNK ® cell line cultured in IMDM-basal medium containing 0.25% human albumin with reducing concentrations of ethanoiamine showed similar ADCC against Ramos cells and direct cytotoxicity against K562 target cells.
• cells cultured in IMDM-basal medium containing 0.125% human albumin showed a drop in direct cytotoxicity against K562 at when ethanoiamine was absent from the medium. Therefore, ethanoiamine may support the functional activity of haNK ® when human albumin is low/ered m the IMDM basal formulation in the absence of insulin, transferrin and sodium selenite supplementation.
• haNK ® cells were evaluated in the current formulation of IMDM-basal medium with and without ethanolamine supplementation, as well as compared with the growth and function of haNK ® cells m reference growth medium.
• a IMDM basal medium containing human AB serum and poloxamer 188 additionally supplemented with 10 mg/L insulin, 5.5 mg/L transferrin, 6.7 pg/L sodium selenite, 2.0 mg/L ethanolamine, 2,0 g/L glucose, 0.125% human albumin will support growth and functional activity of haNK ® cells.
• ethanolamine enhanced the functional activity of haNK ® cells in the presence of 0.125% human albumin in IMDM basal medium supplemented with human AB serum, poloxamer 188 and 2.0 g/L glucose.
• the present study sought to confirm that the IMDM-based formulation containing human AB serum and poloxamer 188 and additionally supplemented with 10 mg/L insulin, 5.5 mg/L transferrin, 6.7 pg/L sodium selenite, 2.0 g/L glucose, 0.125% human albumin would support the growth and function of haNK ® cells with and without additional supplementation of 2.0 mg/L of ethanolamine.
• haNK ® cells were cultured in each medium formulation for five growth cycles of 3 to 4 days in T75 tissue culture flasks. For each growth cycle, the cells were grown from ⁇ 0.3 x 10 6 cells/mL to A3.8-1.2 x 10 6 cells/mL per growth cycle.
• ADCC antibody-dependent cellular cytotoxicity
• Sodium Selenite is present in the IMDM basal media formulation at 17 pg/L.
• the media was supplemented with 6.7 pg/L Sodium Selenite to give a final concentration of 23.7 pg/L Sodium Selenite.
• Glucose is present in the IMDM basal media formulation at 4.5 g/L.
• the media was supplemented with 2 g/L glucose to give a final concentration of 6.5 g/L glucose.
• both basal medium were supplemented with human AB serum and poioxamer 188.
• Sodium selenite is present in the IMDM basal medium formulation at 17 pg/L.
• the medium was supplemented with 6.7 pg/L sodium selenite to give a final concentration of
• Glucose is present in the IMDM basal medium formulation at 4.5 g/L.
• the medium was supplemented with 2.0 g/L glucose to give a final concentration of 6.5 g/L glucose.
• Average ⁇ standard deviation doubling time was calculated for five growth cycles from viable ceil density (VCD) of the liaNK ® cell culture determined at the start and end of each growth cycle using an NC200 automated cell counter using the following equation: Ln2/(Ln (VCD End of Cycle/VCD Start of
• Average ⁇ standard deviation viability was calculated for five growth cycles from the cell viability of the aNKTM cell cultures determined at the start and end of each growth cycle using an NC200 automated cell counter.
• ADCC against Ramos cells and direct cytotoxicity against K562 cells were determined using Calcein AM release assays following standard methods
• VCD viable cell density
• Average ⁇ standard deviation viability was calculated for five growth cycles from the cell viability of the haNK ® cell cultures determined at the start and end of each growth cycle using an NC-200TM automated cell counter.
• ADCC agamst Ramos cells and direct cytotoxicity against K562 cells were determined using Calcein AM release assays following standard methods.
• HaNK ® cells exhibited a similar growth rate and viability to haNK ® cells grown in reference growth medium when grown in IMDM basal medium containing supplements with (Group B versus Group A) and without (Group C versus Group A) ethanolamine.
• the direct cytotoxic function of the haNK ® cell line was marginally impaired at Cycle 3 and more profoundly impaired at Cycle 5 in the absence of ethanolamine supplementation (Table 12 and Figure 1), indicating ethanolamine is important to maintain the functional activity in the current IMDM based medium formulation.
• haNK ® cells were grown for five growth cycles in the indicated medium and were assessed for their direct cytotoxic activity against K562 cells in a Calcein AM-based cytotoxicit assay at the indicated effector to target ratios.
• Direct cytotoxicity function of the haNK ® cell line in medium supplemented with ethanolamine (“LMDM + Insulin, Transferrin, Selenium, Ethanolamine”) was significantly higher than the medium that is not supplemented (“IMDM + Insulin, Transferrin, Selenium”).
• IMDM basal medium containing human AB serum and poloxamer 188 and additionally supplemented with 10 mg/L insulin, 5.5 mg/L transferrin, 6.7 pg/L sodium selenite, 2.0g/L glucose, 0.125% human albumin and 2.0 mg/L ethanolamine supports an equivalent growth rate, viability, and functional activity of haNK ® cells compared to haNK ® cells grown in haNK ® reference growth medium.
• the results also indicate that supplementation with ethanolamine enhances the functional activity of haNK ® cells grown in the IMDM basal medium formulation.
• haNK ® ce!ls grown in the medium compositions as described in Table 13 w3 ⁇ 4re assayed for the surface expression of various markers for NK-92 ® cells, CDS 6, CDS, CD54, CD 16, NKG2D, and NKp30 by flow cytometry. The percentages of cell surface marker expression of the markers are shown m Table 13.
• haNK ® Ref. are haNK ® cells that were grown in refrence growth media containing human AB serum and Poloxamer 188.
• haNK ® ceils were grown in the NK- 92 ® culture media, described in Table 14 and 15, at large scale culture volumes equal to and greater than 5 liters in a bioreactor.
• the cells were grown from ⁇ 0.3 x 10 ⁇ cells/mL to ⁇ 0.8- 1.2 x 10 6 celis/mL per growth cycle and growth rate (doubling time) and viability was determined by automated cell counting at the time of seeding and harvest.
• Functional activity was measured as antibody dependent cellular cytotoxicity (ADCC) against Ramos cells in the presence of 1 pg Rituxan using Calcein AM release assays following standard methods during, at the of production and following cyropresevation and thaw.
• the surface expression of various markers for NK-92 cells, CD56, CD3, CD 54, CD 16, NKG2D, NKp30 was measured using flow cytometry. The percentages of cell surface marker expression of the markers are shown in Table 15 below.
• NK-92 ® Culture media were supplemented with 5% Human AB Serum and 0.05% Poloxamer 188 in addition to the supplement formula shown in the table.
• VCD viable cell density
• J Average viability was measured at the specified process stage using an NC200 automated cell counter.
• SEQ ID NO: 1 High Affinity Variant Immunoglobulin Gamma Fc Region Receptor III-A nucleic acid sequence (full length form).
• ATGTGGCA GCTGCTGCTG CCTACAGCTC

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